Carcinoma homing peptide (chp), its analogs, and methods of using

ABSTRACT

A mini-peptide and its analogs have been found to target gene products to tumors. The peptide, named Carcinoma Homing Peptide (CHP), increased the tumor accumulation of the reporter gene products in five independent tumor models, including one human xenogeneic model. A CHP-IL-12 fusion gene was also developed using CHP and the p40 subunit of IL-12. The product from CHP-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment. In three tumor models. CHP-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for CHP was identified as vimentin. CHP can be used to improve the efficacy and safety of targeted cancer treatments.

The benefit of the Feb. 11, 2011 filing date of the U.S. provisionalpatent application Ser. No. 61/441,914 is claimed under 35 U.S. §119(e).

This invention was made with government support under grant number R01CA120895 awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

TECHNICAL FIELD

This invention pertains to a carcinoma homing peptide and its analogs,compounds, and methods that target tumors, and methods to use thesepeptides including targeting, decreasing the size of, inhibiting growthof, and identification of mammalian tumors, such as breastadenocarcinoma, squamous cell carcinoma, and colon carcinoma.

BACKGROUND ART

The cytokine, interleukin 12 (IL-12), discovered by Giorgio Trinchieriin 1989 [1], bridges the innate and adaptive immune responses byinducing interferon-γ (IFN-γ) production primarily from natural killerand T cells. Cancer therapy with IL-12 exploits its natural immunefunctions to polarize T cells to the T_(h)1 phenotype, boost effector Tcells, downregulate angiogenesis, remodel the extracellular matrix, andalter the levels of immune suppressive cytokines [2]. Due to theseactivities, IL-12 is one of the most promising cytokines forimmunomodulatory cancer therapy.

The initial clinical trials with IL-12 resulted in grave toxicitiesincluding deaths, which severely downgraded the reputation and potentialapplication of this effective cytokine. In reality, most anticancerdrugs or biological modalities are associated with systemic toxicity. Itis desirable to decrease this toxicity to effectively and safely treatthe extremely high numbers of cancer patients [2].

A popular strategy for sequestering the effects of cytokine therapies inthe tumor environment is targeting cellular markers that are upregulatedexclusively in the tumor cells or the tumor microenvironment. Indeed,conjugating IL-12 to tumor-specific antibodies, such as L19 [3] and HER2[4], and tumor vasculature-specific peptides, such as ROD [5] and CNGRC(SEQ ID NO:9) [6], improves the efficacy of treatments; however, thenecessarily high frequency of administrations of recombinant cytokinesincreases the immunogenicity, toxicity, and cost of treatments. A genetherapy approach would reduce these limitations.

Intratumoral IL-12 gene therapy is able to eradicate 40% of tumors in amurine squamous cell carcinoma model (SCCVII) while systemic deliveryvia intramuscular administration fails to eradicate any tumors [7];however, direct injection into tumor sites is rarely availablenoninvasively or post-surgically. Several methods have been developed totarget the IL-12 effect to the tumor after systemic delivery. Forexample, modifying viral vectors with tissue specific gene promoterssuch as the CALC-I promoter [8], capsid-expressed tumor-specificpeptides [9], and polyethylene glycol or other nanoparticles [10, 11]increases tumor specific expression and decreases systemic expression;however, the fenestrated vasculature of the tumor environment allows forthe gene products to leak out of the tumor environment leading tosystemic toxicities [12]. Therefore, a gene product that can interactwith and remain in the tumor environment will increase the level oftherapeutic efficacy and decrease systemic toxicity.

Tumor targeting can be achieved via the screening of various librariesto select tumor-targeted peptides, DNA/RNA aptamers, antibodies, etc;however, the only mechanism that can be used for homing gene productsfrom systemically injected genes will be tumor-targeted mini-peptidesencoding DNA. The small size of these peptides eliminates the concern ofimmunogenicity, and reduces the effect on the biological function of thegene product, though some minipeptidies may boost or inhibit genefunction [20]. The tiny peptide encoding DNA sequences can be easilyfused with any therapeutic gene. Finally, these peptides can complementexisting tumor targeting approaches such as transcriptional targeting[8], translational targeting [21], and targeted delivery [3-6].

Currently, most tumor-targeting strategies are based on extremelyspecific interactions, and the ability to target the tumor environmentis constrained to a single cell type or specific type of tumor. Proteinsare conjugated with polyunsaturated fatty acids, monoclonal antibodies,folic acid, peptides, and several other chemicals to increase thetumor-targeted ability of the therapeutic protein. Other tumor targetingpeptides can deliver small molecules with only one copy for eachsmall-molecule payload but require multiple copies of the peptide totarget larger molecules such as a full length cytokine [24].

DISCLOSURE OF THE INVENTION

We have discovered a new tumor targeting peptide, VNTANST (SEQ ID NO:1),and its analogs. A DNA fragment encoding VNTANST (SEQ ID NO:1) wasinserted directly before the stop codon of the p40 subunit of the IL-12encoding sequence in plasmid DNA. Transfection of this plasmid DNA viaintramuscular (i.m.) electroporation (EP) into muscle tissue distal fromthe tumor site inhibited tumor growth and extended survival in multipletumor models and two mouse strains and reduced lung metastasis in aspontaneous metastatic model. Due to this broad targeting nature and tosimplify the description, the peptide VNTANST (SEQ ID NO:1) was renamedthe Carcinoma Homing Peptide (CHP). We discovered that the linearpeptide VNTANST (SEQ ID NO:1) increased the tumor accumulation of thereporter gene products in five independent tumor models including onehuman xenogeneic model. The product from VNTANST-IL-12 fusion genetherapy increased accumulation of IL12 in the tumor environment, and inthree tumor models, VNTANST-IL-12 gene therapy inhibited distal tumorgrowth. In a spontaneous lung metastasis model, inhibition of metastatictumor growth was improved compared to wild-type (wt) IL-12 gene therapy,and in a squamous cell carcinoma model, toxic liver lesions werereduced. The receptor for VNTANST (SEQ ID NO:1) was identified asvimentin, which is localized on the cell surface of tumor cells but noton normal cells. Vimentin expression in tumors is associated with theepithelial to mesenchymal transition and increased malignancy andmetastasis in tumors. Lastly, this gene product-targeted approachminimized the risk of IL-12-induced toxicity. These results show thepromise of using VNTANST (SEQ ID NO:1) to as a homing peptide to targettherapeutic compounds to tumor cells, for example, to improve deliveryof IL-12 treatments.

We have developed a fully functional tumor targeting IL-12 geneconstruct that can be delivered systemically for treating distallylocated neoplastic diseases. We have administered the peptide CHP-IL-12by direct intravenous injection, and have directly injected the geneconstruct into tissue followed by electroporation. Insertingpeptide-encoding sequences directly prior to the stop codon in the p40gene of an IL-12 plasmid did not interfere with transcription,translation, post-translational modifications, or therapeuticfunctionality of the IL-12 gene product. Also, CHP maintained itstumor-targeting ability as seen in IL-12^(−/−) mice and increased thetherapeutic efficacy of systemic IL-12 gene-therapy treatments whiledecreasing liver toxicity. In fact, CHP-IL-12 may home or target thetumor better than CHP alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the peptide-SEAP (secreted alkaline phosphatase)constructs with insertion of the peptide-coding sequence directly beforethe stop codon (arrow). CMV shows the location of the cytomegaloviruspromoter; IVS shows the location of the intron; pA shows the location ofthe bovine growth hormone polyadenylation signal; SEAP shows thelocation of the secreted alkaline phosphatase-coding sequence; STOPshows the location of the stop codon.

FIG. 1B shows TIS SEAP (ratio of the SEAP activity between tumors andserum) levels 72 hours after i.m. EP of several peptide-SEAP plasmidDNAs in syngeneic CT26 (n=3), SCCVII (n=4), AT84 (n=4), and 4T1 (n=4)tumor-bearing mice, as well as xenogeneic MCF7 (n=4) tumor-bearing mice.

FIG. 1C shows DAB (diaminobenzidine) staining of tumor tissues fromCHP-biotin treated mice counterstained with either hematoxylin (left) oreosin (right). The bottom images are larger versions of the areas withinthe white squares. Bar=100 μm in the top panels and bar=200 μm in thebottom panels.

FIG. 1D shows DAB staining of tumor tissues from Control-peptide-biotintreated mice counterstained with either hematoxylin (left) or eosin(right). The bottom images are larger versions of the areas within thewhite squares. Bar=100 μm in the top panels and bar=200 μm in the bottompanels.

FIG. 2A depicts the CHP-IL-12 construct with insertion of the CHP-codingsequence directly before the stop codon in the p40 subunit of IL-12(arrow). CMV shows the location of the cytomegalovirus promoter; IVSshows the location of the intron; SEAP shows the location of theSEAP-coding sequence; STOP shows the location of the stop codon; and, pAshows the location of the bovine growth hormone polyadenylation signal.

FIG. 2B shows expression of IL-12 after in vitro transfection of 4T1cells with control, wtIL-12, CDGRC-IL-12, and CHP-IL-12 (n=3).

FIG. 2C shows induction of IFN-γ from splenocytes after transfer ofcondition medium containing Control, wtIL-12, CDGRC-II-12, or CHP-IL-12gene products.

FIG. 2D shows IL-12 accumulation in tumor-bearing IL-12^(−/−) micetreated with CHP-IL-12 or wtIL-12 determined via an IL-12p70 ELISA.Columns represent the wtIL-12-normalized level of IL-12/protein (pg/mg)in tumor per IL-12/protein (pg/mg) in kidneys, livers, and spleens andIL-12 μg/mL serum (n=4). Error bars represent the standard error of themean (SEM) (*represent p<0.05 compared to all groups).

FIG. 3A shows tumor growth following treatments with CHP-IL-12, wtIL-12,and control plasmid DNA in 4T1 tumor-bearing balb/c mice (n=5;*represents p<0.05 at day 30 and p<0.001 from day 33 until day 42compared to wtIL-12 plasmid DNA and p<0.01 at day 21 and p<0.001 fromday 24 to day 33 compared to control plasmid DNA).

FIG. 3B shows metastatic nodules in the lungs of 4T1 tumor-bearingbalb/c mice (n=5) treated with CHP-IL-12, wtIL-12, and control plasmidDNA and sacrificed 17 days after the second treatment (*representsp<0.05 compared to wtIL-12 plasmid DNA; # represents p<0.001 compared tocontrol plasmid DNA).

FIG. 3C shows Kaplan-Meier survival analysis of the 4T1 tumor-bearingbalb/c mice treated with CHP-IL-12, wtIL-12, and control plasmid DNA(*represents p<0.05 compared to wtIL-12 plasmid DNA; # representsp<0.001 compared to control plasmid DNA).

FIG. 3D shows tumor growth following treatments with CHP-IL-12, wtIL-12,and control plasmid DNA in SCCVII tumor-bearing C3H mice (n=5;*represents p<0.05 on days 17 and 20 compared to wtIL-12 plasmid DNA andcontrol plasmid DNA).

FIG. 3E shows Kaplan-Meier survival analysis of the SCCVII tumor-bearingC3H mice treated with CHP-IL-12, wtIL-12, and control plasmid DNA(*represents p<0.05 compared to wtIL-12 and control plasmid DNA).

FIG. 3F shows tumor growth following treatments with CHP-IL-12, wtIL-12,and control plasmid DNA in CT26 tumor-bearing balb/c mice (n=5;*represents p<0.05 compared to wtIL-12 plasmid DNA, n=4, on day 25, andcontrol plasmid DNA, n=3, on days 19 through 25). Black arrows representtreatments, and error bars represent SEM.

FIG. 4A shows fluorescence-activated cell sorting (FACS) analysis oftumor infiltrating cells isolated from SCCVII tumors from C3H micefollowing intravenous (i.v.) injection of Control, wtIL-12, orCHP-II-12, with or without depletion of vimentin with a co-injection ofpurified polyclonal goat anti-vimentin (100 μg) in the same i.v.injection as the peptide-biotin collected 7 days after the secondtreatment. The top right quadrant of the dot plot representation ofcells gated for CD11c⁺ represents activated DC (CD81^(hi)).

FIG. 4B shows tumor-specific cytotoxic T lymphocyte (CTL) activity fromwtIL-12 and CHP-IL-12 fusion gene plasmid DNA treated mice bearingorthotopic EMT6 (a transplantable mouse mammary tumor cell line) tumorscollected (*represents p<0.05).

FIG. 4C shows serum IFN-γ levels from 4T1-tumor bearing Balb/c 3 daysafter treatments with CHP-IL-12, wtIL-12, and control plasmid. Errorbars represent SEM.

FIG. 5A shows SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) analysis of potential receptors for CHP isolated viaaffinity chromatography of a pool of cell-surface proteins isolated fromSCCVII cells. The only distinct band (arrow) was located in the secondfraction, and mass spectrometry identified this band as vimentin. “BSA”represents bovine serum albumin.

FIG. 5B shows the interaction of CHP-biotin with recombinantvimentin-GST (Vimentin), GST, and coating buffer only (control) coatedwells of a polystyrene plate (n=6; *represents p<0.001 compared to bothGST and Control, errors bars represent SEM).

FIG. 5C shows Western blot analysis of vimentin expression in an SCCVIItumor (1) and heart (2), lung (3), liver (4), kidney (5), spleen (6),and serum (7) from SCCVII-tumor bearing C3H mice. “GAPDH” representsglyceraldehyde 3-phosphate dehydrogenase.

FIG. 5D shows Western blot analysis of vimentin expression in in vitroand ex vivo tumor samples from SCCVII, CT26, 4T-1, and B16F10 tumors.

FIG. 5E shows accumulation of peptide-biotin in syngeneic SCCVII tumorbearing C3H mice following i.v. injection of either Control-biotin (topleft and right) or CHP-biotin (bottom left and right), with (top andbottom right) or without (top and bottom left) depletion of vimentinwith a co-injection of purified polyclonal goat anti-vimentin (100 μg)in the same i.v. injection as the peptide-biotin.

FIG. 6A shows the number of SCCVII tumor-bearing C3H mice with toxiclesions on the liver following two treatments of 1 μg (2×1 μg), 2 μg(2×2 μg), or 10 μg (2×10 μg) or three treatments of 2 μg (3×2 μg) ofwtIL-12 or CHP-IL-12 (n=12).

FIG. 6B shows a representative image of a normal liver area from theSCCVII tumor-bearing C3H mice. Scale bar represents 50 μm.

FIG. 6C shows a representative image of a toxic lesion from the SCCVIItumor-bearing C3H mice. Scale bar represents 50 μm.

FIG. 6D shows levels of alanine transaminase (ALT), a key indicator ofliver function, for both plasmid DNA treatments (wtIL-12 and CHP-IL-12)at all DNA levels and difference time points.

FIG. 7A shows SEAP activities in the tumors of the same CT26-tumorbearing mice used in FIG. 1B after peptide-SEAP plasmid DNAintramuscular electroporation of several peptides.

FIG. 7B shows SEAP activities in the serum of the same CT26-tumorbearing mice used in FIG. 1B after peptide-SEAP plasmid DNAintramuscular electroporation of several peptides.

FIG. 8 shows sections from the hearts, lungs, livers, kidneys, andspleens from the same mice in FIG. 2B, FIG. 2C and FIG. 2D,counterstained with eosin only.

FIG. 9 shows the level of CHP-specific IgG from EMT6-tumor bearingBalb/c mice treated with wtIL-12 or CHP-IL-12 gene therapy as determinedvia binding to wells of a microwell plate coated with coating bufferonly, control peptide or CHP peptide (n=3).

FIG. 10 shows the activity of CHP-SEAP when bound to vimentin. Theinduction of IFN-γ from splenocytes by CHP-IL12 and wtIL12 was comparedwhen in the presence of vimentin or BSA. Error bars represent SEM andn=3.

FIG. 11 shows the tumor volume in SCCVII tumor-bearing C3H mice atvarious days after inoculation with various gene constructs, eachcomprising the named peptide added to the p40 subunit of IL-12 prior tothe stop codon.

MODES FOR CARRYING OUT THE INVENTION

Tumor targeting can be achieved via the screening of various librariesto select tumor-targeted peptides, DNA/RNA aptamers, antibodies, andother known strategies. However, the only mechanism that can be used forhoming gene products from systemically injected genes is the use of DNAsequences encoding for tumor-targeted mini-peptides. The small size ofthese peptides eliminates the concern of immunogenicity, as shown below,and reduces the effect on the biological function of the gene product,though some mini-peptides may boost or inhibit gene function [20]. Thepeptide-encoding DNA sequences can be easily fused with any therapeuticgene. Finally, the use of the mini-peptides can complement existingtumor targeting approaches such as transcriptional targeting,translational targeting, and targeted delivery].

We have discovered a tumor-targeting 7-amino-acid peptide, carcinomahoming peptide (“CHP,” amino acid sequence of VNTANST (SEQ ID NO: 1)).The peptide VNTANST (SEQ ID NO:1) was previously reported to targetnormal lungs when present on the surface of virus particles [14]. Wehave shown that CHP was more effective than the known cyclictumor-homing peptides such as CNGRC (SEQ ID NO:9) and RGD4C fortargeting to tumors, which rely on disulfide bonds to maintain thecyclic structure of the targeting peptides.

Other tumor targeting peptides have been shown to deliver smallmolecules with only one copy for each small-molecule payload but requiremultiple copies of the peptide to target larger molecules such as a fulllength cytokine [24]. We have shown that fusion of a single copy ofCHP-encoding DNA (gtcaacacggctaactcgaca (SEQ ID NO:2)) with the p40subunit of IL-12 boosted the accumulation of IL-12 in tumors, suggestingone copy of CHP is sufficient to carry one copy of IL-12 to the tumorsite.

Currently, most tumor-targeting strategies are based on extremelyspecific interactions, and the ability to target the tumor environmentis constrained to a single cell type or specific type of tumor. We haveshown, as discussed below, that CHP increased the efficacy of IL-12 genetherapy to inhibit tumor growth in the three tumor cell lines (i.e.,breast adenocarcinoma, squamous cell carcinoma, and colon carcinoma),and in two different mouse strains. In addition, CHP-IL-12 extendedsurvival more than wtIL-12 treatments in both the breast adenocarcinomaand squamous cell carcinoma cell lines. Similarly, CHP-IL-12 treatmentsinhibited the development of spontaneous lung metastasis, which is theprimary killer of cancer patients. This increase in anti-tumor responsewas associated with increases in both tumor-specific cytotoxic Tlymphocyte (CTL) activity and IL-12 accumulation in tumors. This resultwas in agreement with the result that intratumoral delivery of IL-12yields better anti-tumor efficacy than systemic delivery [7]. Thediscovery of CHP is important since it will allow for systemic deliveryto target IL-12 to tumors without the need of intratumoral delivery,which is not realistic for treating internal tumors, metastatic tumors,and residual tumor cells after standard therapy.

We also identified vimentin as a cell receptor for CHP. Vimentin is anintermediate filament protein conventionally regarded as anintracellular structural protein in cells of mesenchymal origin such asfibroblasts, chondrocytes, and macrophages [15]. Vimentin expression hasbeen reported to be increased in several tumor models, including humanprostate, colon [17], hepatocellular [16], and gemcitabine-resistantpancreatic cancers[19], and the tumor stromal cells in human colorectaltumors [18]. The upregulation of vimentin is associated with theepithelial-to-mesenchymal transition (EMT), which is important formotility as well as metastasis in several tumors. In addition, vimentinwas recently discovered to be expressed on the cell surface of tumorcells [25] and epithelial cells during angiogenesis [26]. Additionally,some human tumor-initiating cells remaining after treatment overexpressvimentin on the tumor cell surface [27]. Another important aspect ofvimentin is the conserved sequences among mouse, rat, dog, and humans[28]. This information along with our result for the tumor/serum SEAPaccumulation in the xenogeneic human tumor model indicates that CHPtargeting will be effective in human treatments.

We also confirmed (as discussed below) that vimentin is expressed atvery low levels in the heart, liver, kidney, spleen, and serum of C3Hmice, yet it is highly expressed in lung tissue. However, since mostgeneral expression of vimentin is intracellular [15, 29, 30], thisexpression should not be a target of CHP. We found that there was noaccumulation of CHP-biotin in the lung sections which supports thistheory. Conversely, as shown below, vimentin is highly expressed inaggressive murine squamous cell carcinoma (SCCVII) tumors in C3H mice,and CHP-biotin accumulated in the SCCVII tumors. Likewise, the tumorcells and corresponding syngeneic tumors both expressed detectablelevels of vimentin. The differences seen between expression in tumorcell lines and the respective tumor tissues was due to the heterogeneousnature and multiple cell types in the tumor microenvironment.

We have developed a fully functional tumor-targeting IL-12 p40 geneconstruct based on CHP that can be delivered systemically for treatingdistally located neoplastic diseases. Inserting peptide-encodingsequences directly prior to the stop codon in the p40 subunit gene of anIL-12 plasmid did not interfere with transcription, translation,post-translational modifications, or therapeutic functionality of theIL-12 gene product. Also, CHP maintained its tumor-targeting ability asseen in IL-12−/− mice and increased the therapeutic efficacy of systemicIL-12 gene-therapy treatments, while decreasing liver toxicity.CHP-IL-12 was found to be more effective in decreasing tumor growth thanother mini-peptides linked to the same p40 subunit of IL-12.

The term “CHP” used herein and in the claims refers to the peptideVNTANST. The term “CHP analogs” is understood to be peptides withconsecutive sequences of 3 or more amino acids from VNTANST (SEQ IDNO:1) and that exhibit a qualitatively similar effect to the unmodifiedVNTANST (SEQ ID NO:1) peptide. Based on the effective size of othermini-peptides, we believe that effective CHP analogs include any threeor greater consecutive amino acid sequence found within the CHPsequence, more preferably any four or greater consecutive amino acidsequence found within the CHP sequence, and most preferable any five orsix consecutive amino acid sequence found within the CHP sequence. Inaddition, any DNA sequence that codes for any of the above VNTANST (SEQID NO:1) sequence or CHP analog sequences can be used for making tumortargeting constructs. In the experiments below, we used the DNA sequenceof gtcaacacggctaactcgaca (SEQ ID NO:2) to encode for CHP, but due to thedegeneracy of the DNA code, any DNA sequence that would code for CHPcould be used. In addition, any DNA sequence that encodes for the CHPanalogs could be used. CHP or CHP analog may be a synthetic orrecombinant peptide. With its specific tumor targeting property, CHPpeptide or CHP analogs or the DNA encoding for CHP or CHP analogs cancarry therapeutic proteins, peptides, drugs, genes, cells, viral ornonviral vectors, bacteria and other modalities into tumor tissues,reducing the toxicity to other organs and increasing the therapeuticefficacy. As a result, a low dose of the peptide or construct may beneeded for treating tumors. CHP or CHP analogs or the corresponding DNAencoding for CHP or CHP analogs can also be used to carry therapeuticagents for prevention or treatment of metastatic tumors. Therapeuticagents are well known in the art (e.g., peptides, chemotherapeuticagents, liposomes, nanoparticles) that can be conjugated to a targetedpeptide for increased accumulation of the therapeutic agent in the tumorenvironment.

CHP and CHP analogs can be used in a variety of applications includingexploratory studies to diagnose tumors or tumor metastasis incombination with image tools, to monitor the effect of treatments incombination with image tools, and to deliver therapeutic agents fortreating metastatic tumors and tumors localized in internal organs aswell as prevent tumor recurrence from residual tumors after standardtherapy. The therapeutic agents to be carried by CHP and CHP analogsinclude anti-tumor drugs, peptides, proteins, genes, cells,viral/nonviral vectors, bacteria and others. For example, the p40subunit of the protein IL-12 was used below. We have made a newconjugate of CHP and the p40 subunit of IL-12. The sequence of this newconstruct is found in Table 1, below. The peptide sequence for CHP-IL-12is SEQ ID NO: 3, and the nucleic acid sequence is SEQ ID NO: 4. Initialwork on conjugating other cytokines to CHP, for example IL-15 and PF4,indicate that some increase in efficacy was seen for IL-15, but that inthese initial tests, no increase in efficacy was seen in CHP-PF4.

CHP and CHP analogs can be administered by methods known in the art. Inour work, we have used both direct injection of the gene construct intotissue followed by electroporation, and have directly injected thepeptide intravenously. As a DNA gene construct, the delivery can be fromvectors which may be derived from viruses or from bacterial plasmids.There are many methods to deliver gene constructs to tumors or targetedtissues. Some examples of the various delivery systems can be found inU.S. Pat. Nos: 5,910,488; 7,192,927; and 7,318,919; whose descriptionsof such delivery systems are hereby incorporated by reference. Inaddition, the vector delivery system may incorporate a promoter sequenceto initiate transcription of the gene construct.

1. A tumor-targeting conjugate comprising an agent conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1.
 2. The tumor-targeting conjugate of claim 1, wherein the agent is a reporter peptide or a protein tag or an antitumor therapeutic agent.
 3. The tumor-targeting conjugate of claim 2, wherein the reporter protein is secreted alkaline phosphatase.
 4. The tumor-targeting conjugate of claim 2, wherein the protein tag is biotin.
 5. The tumor-targeting conjugate of claim 2, wherein the anti-tumor therapeutic agent is a cytokine.
 6. A composition comprising the tumor targeting conjugate of claim
 1. 7. The tumor-targeting conjugate of claim 1, prepared by a method comprising conjugating the agent to the carcinoma homing peptide (CHP) consisting of SEQ ID NO:1.
 8. A method for targeting an agent to a vimentin-expressing cell comprising contacting the vimentin-expressing cell with a tumor targeting conjugate comprising the agent conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1, wherein the CHP binds to the vimentin.
 9. The method of claim 8, wherein the agent is an anti-tumor therapeutic agent.
 10. The method of claim 9, wherein the anti-tumor therapeutic agent is a cytokine.
 11. The method of claim 9, wherein the cytokine is interleukin
 12. 12. The method of claim 9, wherein the anti-tumor therapeutic agent is a p40 subunit of interleukin
 12. 13. The method of claim 8, wherein the tumor targeting conjugate comprises an amino acid sequence of SEQ ID NO:3.
 14. The method of claim 8, wherein the tumor targeting conjugate is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:2.
 15. A method of determining the presence of a vimentin protein on a surface of a cell comprising: contacting the cell with a tumor targeting conjugate comprising a peptide conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1 wherein the tumor targeting conjugate binds to the vimentin protein; and assaying the cell for the presence of a bound tumor targeting conjugate such that the presence of the bound tumor targeting conjugate correlates with the presence of the vimentin protein on the cell.
 16. The method of claim 15, wherein the first peptide is a reporter peptide or a protein tag.
 17. The method of claim 16, wherein the reporter peptide is secreted alkaline phosphatase.
 18. The method of claim 16, wherein the protein tag is biotin.
 19. The method of claim 15, wherein the cell is from a biological sample from a mammal.
 20. The method of claim 19, further comprising determining an amount of the vimentin protein present on the cell; and comparing the amount of the vimentin protein present on the cell to a control amount of vimentin protein present on a control cell, wherein the presence of an increased amount of the vimentin protein present on the cell as compared with the control amount indicates that the cell is cancerous. 